DESCRIPTION (Applicant's Description): RNA interference (RNAi) is the surprising ability of double-stranded RNA (dsRNA) when introduced into an animal to direct the specific degradation of a corresponding mRNA. In the last two years, RNAi has been identified in many animals, including flies, worms, and mice, and has provided a new tool for studying gene function and for functional genomics. Evidence is mounting that the cellular function of RNAi is to maintain the integrity of the genome by suppressing transposon "jumping." The RNAi machinery may also serve to defend cells against viral infection and perhaps even to regulate gene expression in the germ line. To begin to understand the mechanism underlying RNAi, we recently developed a cell-free system from Drosophila embryos that recapitulates many of the features of RNAi. Using this in vitro system, we have begun a molecular analysis of RNAi. In this proposal, our specific goals are (1) to understand how the sequence of the dsRNA determines the sites of cleavage on the target mRNA; (2) to discover the enzymatic mechanism by which the dsRNA is processed to create "guide RNAs" that direct cleavage of the target mRNA; (3) to determine the role of ATP in the RNAi pathway; and (4) to identify the proteins that compose the RNAi machinery. These studies promise to help us better understand the mechanism by which the RNAi machinery recognizes and processes dsRNA and then uses the information in the dsRNA to target a specific mRNA for destruction. Our experiments may also help to decipher the rules for selecting potent dsRNAs for silencing specific gene expression and in designing collections of dsRNAs for functional genomic studies. Finally, since a detailed understanding of RNAi is a prerequisite for developing dsRNA analogs that induce RNAi in mammals but do not provoke non-specific anti-viral responses, our work may ultimately speed the development of dsRNA-based therapies for human diseases.